Interrogating multi-featured arrays

ABSTRACT

A method, apparatus for executing the method, and computer program products for use in such an apparatus. The method includes scanning an interrogating light across multiple sites on an array package including an addressable array of multiple features of different moieties, which scanned sites include multiple array features. Signals from respective scanned sites emitted in response to the interrogating light are detected. The interrogating light power is altered for a first site on the array package during the array scan, based on location of the first site or on a determination that the emitted signal from the first site will be outside a predetermined value absent the altering (which allows for protecting a detector against expected overly bright sites), or is altered during the array scan based on the detected interrogating light power (which allows for compensating for light source drift during an array scan).

FIELD OF THE INVENTION

This invention relates to arrays, particularly biopolymer arrays such asDNA arrays, which are useful in diagnostic, screening, gene expressionanalysis, and other applications.

BACKGROUND OF THE INVENTION

Polynucleotide arrays (such as DNA or RNA arrays), are known and areused, for example, as diagnostic or screening tools. Such arrays includefeatures (sometimes referenced as spots or regions) of usually differentsequence polynucleotides arranged in a predetermined configuration on asubstrate. The array is “addressable” in that different features havedifferent predetermined locations (“addresses”) on a substrate carryingthe array.

Biopolymer arrays can be fabricated using in situ synthesis methods ordeposition of the previously obtained biopolymers. The in situ synthesismethods include those described in U.S. Pat. No. 5,449,754 forsynthesizing peptide arrays, as well as WO 98/41531 and the referencescited therein for synthesizing polynucleotides (specifically, DNA). Insitu methods also include photolithographic techniques such asdescribed, for example, in WO 91/07087, WO 92/10587, WO 92/10588, andU.S. Pat. No. 5,143,854. The deposition methods basically involvedepositing biopolymers at predetermined locations on a substrate whichare suitably activated such that the biopolymers can link thereto.Biopolymers of different sequence may be deposited at different featurelocations on the substrate to yield the completed array. Washing orother additional steps may also be used. Procedures known in the art fordeposition of polynucleotides, particularly DNA such as whole oligomersor cDNA, are described, for example, in U.S. Pat. No. 5,807,522(touching drop dispensers to a substrate), and in PCT publications WO95/25116 and WO 98/41531, and elsewhere (use of an ink jet type head tofire drops onto the substrate).

In array fabrication, the quantities of DNA available for the array areusually very small and expensive. Sample quantities available fortesting are usually also very small and it is therefore desirable tosimultaneously test the same sample against a large number of differentprobes on an array. These conditions require the manufacture and use ofarrays with large numbers of very small, closely spaced features.

The arrays, when exposed to a sample, will exhibit a binding pattern.The array can be interrogated by observing this binding pattern by, forexample, labeling all polynucleotide targets (for example, DNA) in thesample with a suitable label (such as a fluorescent compound), scanningan interrogating light across the array and accurately observing thefluorescent signal from the different features of the array. Assumingthat the different sequence polynucleotides were correctly deposited inaccordance with the predetermined configuration, then the observedbinding pattern will be indicative of the presence and/or concentrationof one or more polynucleotide components of the sample. Peptide arrayscan be used in a similar manner. Techniques for scanning arrays aredescribed, for example, in U.S. Pat. No. 5,763,870 and U.S. Pat. No.5,945,679. However, the signals detected from respective featuresemitted in response to the interrogating light, may be other thanfluorescence from a fluorescent label. For example, the signals may befluorescence polarization, reflectance, or scattering, as described inU.S. Pat. No. 5,721,435.

Array scanners typically use a laser as an interrogating light source,which is scanned over the array features. Particularly in array scannersused for DNA sequencing or gene expression studies, a detector(typically a fluorescence detector) with a very high light sensitivityis normally desirable to achieve maximum signal-to-noise in detectinghybridized molecules. At present, photomultiplier tubes (“PMTs”) arestill the detector of choice although charge coupled devices (“CCDs”)can also be used. PMTs are typically used for temporally sequentialscanning of array features, while CCDs permit scanning many features inparallel.

Laser output power in such array scanners may tend to drift over time.As described in U.S. Pat. No. 5,763,870, it is known to provide anintegral power regulation sensor for a laser which is used to monitorlaser output power. The power sensors (that is, laser light illuminancesensors) are connected to current-regulating circuitry that varies thesupply current to the laser and responds to changes in output power. Forgas lasers, the power sensors may be mounted internally and a beamsplitter redirects a portion of the output beam energy to the powersensor, which may be a photodiode. For semiconductor lasers, the powersensor may be formed on the same substrate as the semiconductor layersthat define the laser device, such as described in U.S. Pat. Nos.5,323,026 and 4,577,320. However, the present invention recognizes thatfor some types of gas lasers it may be difficult to reliably switchbetween two power levels quickly, and that for diode lasers changing thelaser power may have the undesired side effect of changing thewavelength.

The present invention further realizes that strong signals may occur inresponse to the interrogating light, either from bright features or fromother components (for example, fluorescence of the glue holding an arraysubstrate in a housing). In PMTs and other detectors (such as CCDs) verystrong signals that are (depending on the type of detector) eitherspatially and/or temporally close to weak signals, may undesirablyaffect the latter. For example, a PMT reading a very bright signal at agiven time, may change its sensitivity for a short time after this, oron a CCD detector very bright pixels may bleed their charge intoadjacent ones. Extremely strong signals may even damage either kind of(normally expensive) detector. In any event, the accurate detection ofsignals from an array being interrogated by the scanner may be in doubtdue to such effects.

The present invention realizes that it would be desirable then, toprovide a technique for scanning an addressable array which allowed forrapid correction in variations in power of an interrogating light. Thepresent invention further realizes that it would be desirable if somemeans were provided during array scanning, to limit exposure of adetector to very strong signals generated by features or other sites inresponse to the interrogating light. Additionally, the present inventionalso realizes that during the typically rapid scanning of an array, sometypes of light sources may not respond sufficiently rapidly to changesin power input (as discussed above) to allow for the corrections of thepresent invention while maintaining high array scanning speeds.

SUMMARY OF THE INVENTION

The present invention then, provides a method for use with anaddressable array of multiple features of different moieties. Thesemoieties may, for example, be polynucleotides (such as DNA or RNA) ofdifferent sequences for different features. In the method, aninterrogating light is scanned across the array. This scanning can beaccomplished, for example, by moving the interrogating light relative tothe array, moving the array relative to the interrogating light, orboth. The interrogating light is generated from a variable opticalattenuator through which light from a light source has passed, and whichoptical attenuator is responsive to a control signal to alter the powerof the interrogating light. Signals from respective features emitted inresponse to the interrogating light, are detected. A power of theinterrogating light from the variable optical attenuator is alsodetected. An attenuator control signal is adjusted to alterinterrogating light power, which adjustment is based on the detectedpower. The power may be detected and altered at any convenient times.For example, the power may be detected and altered during the scanningstep (such as during a transition of the interrogating light from onerow to another in the case where the array includes rows of featureswhich are scanned row by row).

In a second aspect, the interrogating light is scanned across multiplesites on an array package which includes the array, and which scannedsites include the features. Signals emitted from respective scannedsites in response to the interrogating light, are detected. Theinterrogating light power is adjusted for a first site (which may or maynot be an array feature) on the array package during the array scanbased on some characteristic of the first site, such as location of thefirst site or a determination that the emitted signal from the firstsite will be outside a predetermined range (for example, a lower orupper limit) absent the altering. For example, the interrogating lightpower may be reduced based on a determination that the emitted signalfrom the first site will exceed a predetermined value, or based onlocation of the first site. If the determination is used this can bebased, for example, on the results of a pre-scan or on the signalemitted from the first site when the interrogating light initiallyilluminates the first site. A pre-scan can include scanning aninterrogating light across multiple sites on the array package using alower sensitivity than that used in the previously mentioned scan(sometimes referenced as the “main scan”). Lower sensitivity can beobtained by using a lower interrogating light power, or with an emittedsignal detector of lowered light sensitivity. Signals emitted inresponse to this interrogating light are detected and the determinationbased on one or more of these signals obtained in the pre-scan. Ifinterrogating light power is altered based on location of the firstfeature, this location can be based on a reading (machine or human) ofan identification associated with the array package (such as anidentification carried on the package).

In the second aspect, any alteration of the interrogating light powercan be made using the results of a previously obtained calibration. Inparticular, the method may also include calibrating the interrogatinglight power versus a control signal, for a light system which providesthe interrogating light of a power which varies in response to thecontrol signal. Optionally, this calibration may be repeated beforescanning and detection for each of multiple array packages. Whilevarious light systems may be used in the second aspect, a light systemwhich includes a light source and an optical attenuator in thearrangement disclosed above, may for example be used.

The present invention further provides apparatus for executing methodsof the present invention. In a first aspect, the apparatus includes thelight source and variable optical attenuator, a scanning system tocontrol scanning, an emitted signal detector, and a power detector todetect the power of the interrogating light. The apparatus also includesa system controller which receives input from, and controls theremainder of, the apparatus as required (including using locationinformation or making determinations, as described above) such that theremainder of the apparatus can execute a method of the invention. Forexample, the system controller may adjust the optical attenuator controlsignal to alter interrogating light power, based on the power detectedby the power detector. Such an apparatus can execute the first aspect ofthe method described above. In an alternative example, the systemcontroller adjusts the interrogating light power for a first site on thearray package during the array scan based on location of the first siteor on a determination that the emitted signal from the first site willbe outside a predetermined range absent alteration. Such an apparatuscan execute the second aspect of the method described above. However,the system controller optionally, and preferably, receives input from,and controls the remainder of, the apparatus as required such that thesame apparatus can execute both a method of the first and second aspectsdescribed above.

In one particular embodiment of the apparatus, the apparatus furtherincludes a reader to machine read an identification carried on the arraypackage. In this case, the system controller may determine the locationof the first site based on the read identification. This determinationcan, for example, be made directly from data contained in the readidentification or retrieved from a local or remote source using theidentification.

The present invention further provides a computer program product foruse in an apparatus of the present invention. Such a computer programproduct includes a computer readable storage medium having a computerprogram stored thereon which, when loaded into a computer of theapparatus, such as the controller, causes it to perform the stepsrequired by the apparatus to execute a method of the present invention.

While the methods and apparatus have been described in connection witharrays of various moieties, such as polynucleotides or DNA, othermoieties can include any chemical moieties such as biopolymers. Also,while the detected signals may particularly be fluorescent emissions inresponse to the interrogating light, other detected signals in responseto the interrogating light can include polarization, reflectance, orscattering, signals. Also, the described methods in the presentapplication can be used to alter the actual scanning pattern of theinterrogating light (rather than, or in addition to altering theinterrogating light power) on the array package, particularly based upona read identification (such as a machine readable identification)carried on the array package. For example, the dimensions of the scannedarea, and/or the number of scan lines and/or scan line spacing may bebased on the read identification (with the required information beingretrieved from the read ID or from a local or remote database).

The method, apparatus, and kits of the present invention can provide anyone or more of the following or other benefits. For example, correctionin the power of an interrogating light can be obtained. Also, exposureof a detector to very strong signals generated by features or othersites in response to the interrogating light, can be limited based onrapid altering of interrogating light power. This can avoid bothdetector damage and detector blinding (temporary, light-induced changeof sensitivity) at high switching speeds with a simple apparatus.Further, alterations of interrogation light power may be obtained whichmay be faster than obtainable from altering power to some types of lightsources, so that high array scanning rates can still be maintained.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described with reference to thedrawings, in which:

FIG. 1 is a perspective view of a substrate carrying a typical array, asmay be used with, or part of, a package of the present invention;

FIG. 2 is an enlarged view of a portion of FIG. 1 showing some of theidentifiable individual regions of a single array of FIG. 1;

FIG. 3 is an enlarged cross-section of a portion of FIG. 2;

FIG. 4 is a front view of an array package in the form of a cartridge;

FIG. 5 illustrates an apparatus of the present invention; and

FIG. 6 is a flowchart illustrating a method of the present invention.

To facilitate understanding, the same reference numerals have been used,where practical, to designate similar elements that are common to theFIGS.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the present application, unless a contrary intention appears,the following terms refer to the indicated characteristics. A“biopolymer” is a polymer of one or more types of repeating units.Biopolymers are typically found in biological systems and particularlyinclude peptides or polynucleotides, as well as such compounds composedof or containing amino acid or nucleotide analogs or non-nucleotidegroups. This includes polynucleotides in which the conventional backbonehas been replaced with a non-naturally occurring or synthetic backbone,and nucleic acids (or synthetic or naturally occurring analogs) in whichone or more of the conventional bases has been replaced with a group(natural or synthetic) capable of participating in Watson-Crick typehydrogen bonding interactions. Polynucleotides include single ormultiple stranded configurations, where one or more of the strands mayor may not be completely aligned with another. A “nucleotide” refers toa sub-unit of a nucleic acid and has a phosphate group, a 5 carbon sugarand a nitrogen containing base, as well as analogs (whether synthetic ornaturally occurring) of such sub-units. For example, a “biopolymer”includes DNA (including cDNA), RNA, oligonucleotides, and PNA and otheroligonucleotides as described in U.S. Pat. No. 5,948,902 and referencescited therein (all of which are incorporated herein by reference),regardless of the source. An “oligonucleotide” generally refers to apolynucleotide of about 10 to 100 nucleotides (or other units) inlength, while a “polynucleotide” includes a nucleotide multimer havingany number of nucleotides. A “biomonomer” references a single unit,which can be linked with the same or other biomonomers to form abiopolymer (for example, a single amino acid or nucleotide with twolinking groups one or both of which may have removable protectinggroups). A biomonomer fluid or biopolymer fluid reference a liquidcontaining either a biomonomer or biopolymer, respectively (typically insolution). An “addressable array” includes any one or two dimensionalarrangement of discrete regions (or “features”) bearing particularmoieties (for example, different polynucleotide sequences) associatedwith that region and positioned at particular predetermined locations onthe substrate (each such location being an “address”). These regions mayor may not be separated by intervening spaces. By one item being“remote” from another, is referenced that the two items are at least indifferent buildings, and may be at least one mile, ten miles, or atleast one hundred miles apart. An array “package” may be the array plusonly a substrate on which the array is deposited, although the packagemay include other features (such as a housing). A “chamber” referencesan enclosed volume (although a chamber may be accessible through one ormore ports). It will also be appreciated that throughout the presentapplication, that words such as “top”, “upper”, and “lower” are used ina relative sense only. “Fluid” is used herein to reference a liquid.“Venting” or “vent” includes the outward flow of a gas or liquid.Reference to a singular item, includes the possibility that there areplural of the same items present. All patents and other cited referencesare incorporated into this application by reference.

Referring first to FIGS. 1-3, a contiguous planar transparent substrate10 carries multiple features 16 disposed across a first surface 11 a ofsubstrate 10 and separated by areas 13. Features 16 are disposed in apattern which defines the array. A second surface 11 b of substrate 10does not carry any features. Substrate 10 may be of any shape althoughthe remainder of the package of the present invention may need to beadapted accordingly. A typical array may contain at least ten features16, or at least 100 features, at least 100,000 features, or more. All ofthe features 16 may be different, or some or all could be the same. Eachfeature carries a predetermined moiety or mixture of moieties which inthe case of FIGS. 1-3 is a polynucleotide having a particular sequence.This is illustrated schematically in FIG. 3 where regions 16 are shownas carrying different polynucleotide sequences. Arrays of FIGS. 1-3 canbe manufactured by in situ or deposition methods as discussed above. Inuse, a feature can detect a polynucleotide of a complementary sequenceby hybridizing to it, such as polynucleotide 18 being detected byfeature 16 a in FIG. 3 (the “*” on polynucleotide 18 indicating a labelsuch as a fluorescent label). Use of arrays to detect particularmoieties in a sample (such as target sequences) are well known.

Referring now to FIG. 4 an array package 30 includes a housing 34 whichhas received substrate 10 adjacent an opening. Substrate 10 is sealed(such as by the use of a suitable adhesive) to housing 34 around amargin 38 with the second surface 11 b facing outward. Housing 34 isconfigured such that housing 34 and substrate 10, define a chamber intowhich features 16 of array 12 face. This chamber is accessible throughresilient septa 42, 50 which define normally closed ports of thechamber. Array package 30 preferably includes an identification (“ID”)54 of the array. The identification 54 may be in the form of a bar codeor some other machine readable code applied during the manufacture ofarray package 30. Identification 54 may itself contain instructions fora scanning apparatus that the interrogating light power for at least afirst site of the sites to be scanned and of specified location on arraypackage 30 should be altered (typically, decreased). These instructionsare typically based on the expectation that the emitted signals fromthose sites will be too bright or that those sites are not of interest(for example, they are off the area covered by the array). The specifiedsites (specified by location on array package 30) can be particular onesof features 16 or can be other sites on array package 30 such as margin38 from which, for example, unduly bright fluorescence from an adhesivemight be expected, or regions off the area covered by the array andhence are not of interest (and hence the instructions describe the areato be scanned). Alternatively, identification 54 may be simply a uniqueseries of characters which is also stored in a local or remote databasein association with the foregoing location information. Such a databasemay be established by the array manufacturer and made accessible to theuser (or provided to them as data on a portable storage medium).

It will be appreciated though, that other array packages may be used.For example, the array package may consist only of the array of features16 on substrate 10 (in which case ID 54 can be applied directly tosubstrate 10). Thus, an array package need not include any housing orclosed chamber.

The components of the embodiments of the package 30 described above, maybe made of any suitable material. For example, housing 34 can be made ofmetal or plastic such as polypropylene, polyethylene oracrylonitrile-butadiene-styrene (“ABS”). Substrate 10 may be of anysuitable material, and is preferably sufficiently transparent to thewavelength of an interrogating and array emitted light, as to allowinterrogation without removal from housing 34. Such transparent andnon-transparent materials include, for flexible substrates: nylon, bothmodified and unmodified, nitrocellulose, polypropylene, and the like.For rigid substrates, specific materials of interest include: glass;fused silica, silicon, plastics (for example, polytetrafluoroethylene,polypropylene, polystyrene, polycarbonate, and blends thereof, and thelike); metals (for example, gold, platinum, and the like). The firstsurface 11 a of substrate 10 may be modified with one or more differentlayers of compounds that serve to modify the properties of the surfacein a desirable manner. Such modification layers, when present, willgenerally range in thickness from a monomolecular thickness to about 1mm, usually from a monomolecular thickness to about 0.1 mm and moreusually from a monomolecular thickness to about 0.001 mm. Modificationlayers of interest include: inorganic and organic layers such as metals,metal oxides, polymers, small organic molecules and the like. Polymericlayers of interest include layers of: peptides, proteins, polynucleicacids or mimetics thereof (for example, peptide nucleic acids and thelike); polysaccharides, phospholipids, polyurethanes, polyesters,polycarbonates, polyureas, polyamides, polyethyleneamines, polyarylenesulfides, polysiloxanes, polyimides, polyacetates, and the like, wherethe polymers may be hetero- or homopolymeric, and may or may not haveseparate functional moieties attached thereto (for example, conjugated),The materials from which substrate 10 and housing 34 (at least theportion facing toward the inside of chamber 36) may be fabricated shouldideally themselves exhibit a low level of binding during hybridizationor other events.

Referring to FIG. 5, an apparatus of the present invention (which may begenerally referenced as an array “scanner”) is illustrated with an arraypackage 30 mounted therein. A light system provides light from a laser100 which passes through an electro-optic modulator (EOM)110 withattached polarizer 120. A control signal in the form of a variablevoltage applied to the EOM 110 by the control unit (CU) 180 changes thepolarization of the exiting light which is thus more or less attenuatedby the polarizer 120. Thus, EOM 110 and polarizer 120 together act as avariable optical attenuator which can alter the power of aninterrogating light spot exiting from the attenuator. A small portion ofthe transmitted light is reflected off a beamsplitter 140 to aphotodiode or other power detector 150 (which therefore acts as aninterrogating light power detector). The resulting signal is fed intothe control unit 180. The remainder of the light transmitted bybeamsplitter 140 is in this case reflected off a dichroic beamsplitter154 and focused onto the array of array package 30 using opticalcomponents in beam focuser/scanner 160. Light emitted from features 16in response to the interrogating light, for example by fluorescence, isimaged, for example, using the same optics in focuser/scanner 160, andpasses through the dichroic beamsplitter 154 and onto a detector (PMT)130. More optical components (not shown) may be used between thedichroic and the PMT (lenses, pinholes, filters, fibers etc.) and thedetector 130 may be of various different types (e.g. a photo-multipliertube (PMT) or a CCD or an avalanche photodiode (APD)). A scanning systemcauses the interrogating light spot to be scanned across multiple siteson an array package 30 received in the apparatus, which sites include atleast the multiple features 16 of the array. In particular the scanningsystem is typically a line by line scanner, scanning the interrogatinglight in a line across the array package 30, then moving(“transitioning”) the interrogating light to begin scanning a next row,scanning that next row, and repeating the foregoing procedure row afterrow. The scanning system can be a beam scanner within beamfocuser/scanner 160 which moves the interrogating light across astationary array package 30, or can be a transporter 190 which movesarray package 30 in relation to a stationary interrogating light beam,or may be a combination of the foregoing (for example, with beamfocuser/scanner 160 scanning the interrogating light spot across a rowof features 16 of the array, and with transporter 190 moving the arrayone row at a time such that beam focuser/scanner 160 can scan successiverows of features 16).

The apparatus of FIG. 5 may further include a reader 170 which reads anidentification 54 from an array package 30. When identification 54 is inthe form of a bar code, reader 170 may be a suitable bar code reader.However, as described below, instead of (or in addition to) reader 170,the apparatus may execute a pre-scan or use initially determined emittedsignal data from sites on array package 30.

A system controller 180 of the apparatus is connected to receive signalsemitted in response to the interrogating light from emitted signaldetector 130, signals indicating detected power from detector 150, andsignals indicating a read identification from reader 170, and to providethe control signal to EOM 110. Controller 180 may also analyze, store,and/or output data relating to emitted signals received from detector130 in a known manner. Controller 180 may include a computer in the formof a programmable digital processor, and include a media reader 182which can read a portable removable media (such as a magnetic or opticaldisk), and a communication module 184 which can communicate over acommunication channel (such as a network, for example the internet or atelephone network) with a remote site (such as a database at whichinformation relating to array package 30 may be stored in associationwith the identification 54). Controller 180 is suitably programmed toexecute all of the steps required by it during operation of theapparatus, as discussed further below. Alternatively, controller 180 maybe any hardware or hardware/software combination which can execute thosesteps.

In one mode of operation, the array in package 30 is typically firstexposed to a liquid sample introduced into the chamber through one ofthe septa 42, 50. The array may then be washed and scanned with a liquid(such as a buffer solution) present in the chamber and in contact withthe array, or it may be dried following washing. Referring in particularto FIG. 6 following a given array package 30 being mounted in theapparatus, reader 170 automatically (or upon operator command) reads(210) array ID 54. Controller 180 can then use this ID 54 to retrieve(220) locations on package 30 at which the interrogating light powershould be altered (typically, reduced). Such information may beretrieved directly from the contents of ID 54 when ID 54 contains suchinformation. Alternatively, ID 54 may be used to retrieve suchinformation from a database containing the ID in association with suchinformation. Such a database may be a local database accessible bycontroller 180 (such as may be contained in a portable storage medium indrive 182 which is associated with package 30, such as by physicalassociation with package 30 when received by the user, or by a suitableidentification), or may be a remote database accessible by controller180 through communication module 184 and a suitable communicationchannel (not shown).

The interrogating light power may be calibrated (230) versus a controlsignal. Specifically, this is done by calibrating EOM 110 before orafter the steps 210, 230. In particular, the transmission of the EOM 110is controlled using a high voltage differential input from controller180. For example, a voltage differential of 296 volts may cause aminimum power output, while a voltage differential of 104 volts maycause a maximum power output. The power as a function of differentialvoltage is roughly sinusoidal with an offset from zero and scaling thatvaries with time and temperature. The voltage differential is thedifference between the voltage at a first terminal A and the voltage atsecond terminal B of EOM 110. For easy transition between high and lowtransmission states, terminal A may be held constant while terminal Bmay be toggled between a high voltage state and ground. For example,terminal A may be set to 296 volts, and terminal B switched betweenground and 192 volts. Other approaches are conceivable. When a scan isinitiated, terminal B is set to ground. Then terminal A is swept from 0to 500 volts. The voltage at terminal A is set to where the output powerreaches a local minimum. After that, terminal B is swept from 0 to 500volts. The power output values for given voltages at terminal B will berecorded by controller 180 (which may include suitable analog/digitalconverter for the output of photodiode detector 150). Controller 180will be looking for a specific slope to set the maximum output power. Ifthe maximum power setting is at a local maximum and the output powerdrops, there is no way of telling which direction it went, and thus howto correct for it. By looking for a specific slope near the peak, asmall power fluctuation can be readily converted into a linear powercorrection.

Next, the scanning system scans in a line across a row of array features16 in the manner already described. During such a row scan, the EOM 110is controlled by controller 180 to alter (for example, reduce orincrease) the interrogation light power for at least a first site of thescanned sites on package 30 as required by location data retrieved basedon ID 54. As already mentioned, the one or more of such sites can befeatures 16, margin 38, or some other site. By “reduce” or “increase” inthis context is referenced that the interrogation power for thespecified site is less, or more, respectively, than for another site orsites (usually other previously interrogated features).

However, the location of such first sites can be determined by meansother than from ID 54 and thus steps 210, 220 can be omitted. Forexample, such site information can be obtained during a pre-scan ofpackage 30 executed before the above described main scan. In such apre-scan the same line-by-line scanning procedure as described above isused, but with the apparatus adjusted for a lower sensitivity than usedin the above described main scan, to identify any sites at which theemitted signal may be outside a predetermined range (typically, anemitted signal which will exceed a predetermined limit for detector 130)when the higher apparatus sensitivity of the main scan is used. Duringthe subsequent main scan controller 180 alters interrogating light powerfor one or more such first sites based on the emitted signals obtainedduring the pre-scan. In another alternative arrangement, interrogatinglight power can be altered dynamically for one or more first sites, withcontroller 180 altering interrogating light power at any site based ondetector 130 detecting an emitted signal from the first site when theinterrogating light initially illuminates the first site during thatsame main scan. For example, feedback may be provided that reduces thelaser power transmitted through the polarizer 120 in such a way that thesignal increases as the square root of the number of moleculesilluminated above a certain threshold rather than proportional to thenumber of molecules as it would otherwise do. However, the finiteresponse time of any feedback system may produce possibly relevantartifacts if rapid signal changes occur (for example, when scanningacross the edge of a feature 16). This can be compensated for byignoring the corresponding pixels in the analysis if needed (wherein apixel is typically a subset of a site).

In another alternative for determining the location of first sites of atype which are regions outside the array area, array package 30 can beprovided with suitable markings positioned just outside the array area.These can be detected by detector 130 and are interpreted by controller180 as indicating that all sites beyond such markings are outside thescan area and hence are sites which need not be scanned.

When the dynamic adjustment of interrogating light power is used, thesignal versus brightness curve becomes very non-linear. However thisneed not be visible in data acquired as the signal is normally digitizedand levels can be chosen such that the analog to digital convertersaturates at levels lower than those at which the protection circuit(controller 180 and EOM 110) starts to reduce the amount of laser lightreaching the sample. The dynamic adjustment has the advantage over thepre-scan of requiring only one scan (for example, when protecting fromimpurities on the array), while the pre-scan approach has the advantageof capturing the entire range of features up to extremely bright ones.Both advantages can be obtained using ID 54 though, in the mannerdescribed above.

Returning to FIG. 6, regardless of the whether the data (244) islocation data obtained based on ID 54 or obtained from a pre-scan, orinitial power data, controller 180 will alter interrogating light powerfor one or more first sites as indicated by such data (244). Whenscanning of one row of features is completed the interrogating lightspot will scan off the array somewhat. By scanning “off” the array inthis context is referenced that there are no more features 16 in thedirection of the line along which the interrogating light spot has beenscanning. If it is determined (250) that there is still one or more rowsof the array to be scanned, controller 150 then detects interrogatinglight power (260) from detector 150 and, when the detected power doesnot equal a predetermined target power (“nominal” power), signals EOM110 so as to adjust (270) the power to the target. Such a procedurecorrects for any drift in output power from laser 100 due to temperatureor other fluctuations. Such a checking and correction is preferably madeduring the relatively longer period of a transition from scanning onerow to another (that is, the period where scanning features of one rowhas ended, until the period where scanning features of another rowbegins). This allows power fluctuations due to the corrections to berestricted to non-critical areas. Further, relatively little drift willlikely occur during the scanning of a given row. Laser 100 and EOM 110operating variations while the light source output is at it's minimumare normally not relevant since they constitute only minor fluctuationsof a quantity (output power at minimum transmission) that is alreadysmall. At the minimum of transmission, the change of transmission vs.voltage is zero to first order (first derivative). Hence it may benecessary to dither the voltage slightly to track the minimum (i.e. thevoltage that results in minimum transmission) as it may drift, forexample as a consequence of temperature changes. In some cases it may bedesirable to have additional power levels at ones disposal (see forexample, the implementation of the square-root function above). In thesecases, one can calibrate the system by measuring, storing and using atable of transmission values versus voltages prior to a scan andpossibly also using the derivatives calculated from these for secondorder corrections (for example, compensating for laser powerfluctuations).

The next row is then scanned (240) with any alterations (242) ininterrogating light power being executed as before. However, it will beappreciated that it is possible to adjust interrogating light power morefrequently than just during the transition from one row to another (forexample, when the scanning interrogating light spot is between features16).

The above cycle is repeated until it is determined (250) that the lastrow of an array in package 30 has been scanned. If it is determined(280) that another array is to be scanned (such as by an operatorpressing a “START” button or by a switch in the array holder detectingmounting of another array), the entire cycle is then repeated. Note thatcalibration (230) of EOM 110 before scanning each array corrects for anydrifts in performance (for example due to temperature variations)between array scans. Further, use of an EOM 110 can allow for more rapidalteration of the interrogating light than may otherwise be possible bysimply controlling power to some types of light sources, such as laser100.

Note that a variety of geometries of the features 16 may be constructedother than the organized rows and columns of the array of FIGS. 1-3. Forexample, features 16 can be arranged in a series of curvilinear rowsacross the substrate surface (for example, a series of concentriccircles or semi-circles of spots), and the like. Even irregulararrangements of features 16 can be used, at least when some means isprovided such that during their use the locations of regions ofparticular characteristics can be determined (for example, a map of theregions is provided to the end user with the array). Furthermore,substrate 10 could carry more than one array 12, arranged in any desiredconfiguration on substrate 10. While substrate 10 is planar andrectangular in form, other shapes could be used with housing 34 beingadjusted accordingly. In many embodiments, substrate 10 will be shapedgenerally as a planar, rectangular solid, having a length in the rangeabout 4 mm to 200 mm, usually about 4 mm to 150 mm, more usually about 4mm to 125 mm; a width in the range about 4 mm to 200 mm, usually about 4mm to 120 mm and more usually about 4 mm to 80 mm; and a thickness inthe range about 0.01 mm to 5.0 mm, usually from about 0.1 mm to 2 mm andmore usually from about 0.2 to 1 mm. However, larger substrates can beused. Less preferably, substrate 10 could have three-dimensional shapewith irregularities in first surface 11 a. In any event, the dimensionsof housing 34 may be adjusted accordingly.

The apparatus of FIG. 5 can be constructed accordingly to scan arraypackages of the described structure.

Various modifications to the particular embodiments described above are,of course, possible. Accordingly, the present invention is not limitedto the particular embodiments described in detail above.

What is claimed is:
 1. A method comprising: (a) scanning aninterrogating light across an addressable array of multiple features ofdifferent moieties, which interrogating light is generated from avariable optical attenuator through which light from a light source haspassed and which optical attenuator is responsive to a control signal toalter the power of the interrogating light; (b) detecting signals fromrespective features emitted in response to the interrogating light; (c)detecting a power of the interrogating light from the variable opticalattenuator; and (d) adjusting the attenuator control signal to alterinterrogating light power, based on the detected power.
 2. A methodaccording to claim 1 wherein the multiple features comprisepolynucleotides of different sequences.
 3. A method according to claim 1wherein the multiple features comprise DNA of different sequences.
 4. Amethod according to claim 1 wherein the detected signals are fluorescentemissions from the features.
 5. A method according to claim 1 whereinthe power is detected and altered during step (a).
 6. A method accordingto claim 5 wherein the interrogating light additionally scans acrosssites other than on the array, and wherein the interrogating light poweris decreased when the interrogating light has scanned off the array. 7.A method according to claim 5 wherein the interrogating light is scannedrow by row across the array features, and wherein the interrogatinglight power is altered during a transition of the interrogating lightfrom one row to another.
 8. A method comprising: (a) scanning aninterrogating light across multiple sites on an array package includingan addressable array of multiple features of different moieties, whichscanned sites include multiple features of the array; (b) detectingsignals from respective scanned sites emitted in response to theinterrogating light; and (c) altering the interrogating light power fora first site on the array package during the array scan based onlocation of the first site; wherein the interrogating light power isreduced based on location of the first site.
 9. A method according toclaim 8 wherein the multiple features comprise polynucleotides ofdifferent sequences.
 10. A method according to claim 8 wherein themultiple features comprise DNA of different sequences.
 11. A methodaccording to claim 8 additionally comprising reading an identificationcarried on the array package and determining the location of the firstsite based on the read identification.
 12. A method according to claim11 wherein the identification is machine read.
 13. A method comprising:(a) scanning an interrogating light across multiple sites on an arraypackage including an addressable array of multiple features of differentmoieties, which scanned sites include multiple features of the array;(b) detecting signals from respective scanned sites emitted in responseto the interrogating light; and (c) altering the interrogating lightpower for a first site on the array package during the array scan basedon location of the first site or on a determination that the emittedsignal from the first site will be outside a predetermined range absentthe altering; the method additionally comprising, prior to steps (a)through (c), performing a pre-scan which includes: scanning aninterrogating light across multiple sites on the array package using alower sensitivity than that used in step (a); and detecting signals fromrespective scanned sites emitted in response to the interrogating light;wherein the interrogating light power is altered based on thedetermination that the emitted signal from the first site will beoutside a predetermined range, which is based on the emitted signalobtained from the first site during the pre-scan.
 14. A methodcomprising: (a) scanning an interrogating light across multiple sites onan array package including an addressable array of multiple features ofdifferent moieties, which scanned sites include multiple features of thearray; (b) detecting signals from respective scanned sites emitted inresponse to the interrogating light; and (c) altering the interrogatinglight power for a first site on the array package during the array scanbased on location of the first site or on a determination that theemitted signal from the first site will be outside a predetermined rangeabsent the altering; wherein the interrogating light power is reducedbased on a determination that the emitted signal from the first sitewill exceed a predetermined value; and wherein the first site is not anarray feature.
 15. An apparatus for interrogating an addressable arrayof multiple features of different moieties, comprising: (a) a lightsource and a variable optical attenuator through which light from thesource passes to provide an interrogating light, and which opticalattenuator is responsive to a control signal to alter the power of theinterrogating light; (b) a scanning system which scans the interrogatinglight across the addressable array; (c) a signal detector to detectsignals from respective features emitted in response to theinterrogating light; (b) a power detector to detect the power of theinterrogating light from the variable optical attenuator; and (c) asystem controller which adjusts the optical attenuator control signal toalter interrogating light power, based on the detected power.
 16. Anapparatus according to claim 15 wherein the power detector and systemcontroller co-operate to detect and adjust interrogating light powerprior to scanning each of multiple arrays.
 17. An apparatus according toclaim 15 wherein the power detector and system controller co-operate todetect and adjust interrogating light power during scanning of theinterrogating light across the array.
 18. An apparatus according toclaim 15 wherein the system controller decreases the power when thescanning system causes the interrogating light to scan off the array.19. An apparatus according to claim 18 wherein the scanning systemcauses the interrogating light to scan row by row across the arrayfeatures, and wherein the power is adjusted during a transition of theinterrogating light from one row to another.
 20. An apparatus forinterrogating an addressable array of multiple features of differentmoieties, carried by an array package, comprising: (a) a light systemwhich provides an interrogating light of variable power in response to acontrol signal; (b) a scanning system which scans the interrogatinglight across multiple sites on the array package, which scanned sitesinclude multiple features of the array; (c) a signal detector whichdetects signals from respective sites emitted in response to theinterrogating light; and (d) a system controller which adjusts theinterrogating light power for a first site on the array package duringthe array scan based on location of the first site.
 21. An apparatusaccording to claim 20 wherein the system controller reduces theinterrogating light power for the first site.
 22. A method according toclaim 20 additionally comprising a reader to machine read anidentification carried on the array package, and wherein the systemcontroller determines the location of the first site based on the readidentification.
 23. An apparatus according to claim 20 wherein the lightsystem includes a light source and an optical attenuator through whichlight from the source passes to provide the interrogating light, andwherein the control signal comprises a signal for the optical attenuatorwhich provides variable attenuation in response to the characteristic ofthe control signal.
 24. An apparatus for interrogating an addressablearray of multiple features of different moieties, carried by an arraypackage, comprising: (a) a light system which provides an interrogatinglight of variable power in response to a control signal; (b) a scanningsystem which scans the interrogating light across multiple sites on thearray package, which scanned sites include multiple features of thearray; (c) a signal detector which detects signals from respective sitesemitted in response to the interrogating light; and (d) a systemcontroller which adjusts the interrogating light power for a first siteon the array package during the array scan based on location of thefirst site or on a determination that the emitted signal from the firstsite will be outside a predetermined range absent alteration; whereinadjustment is based on the determination by the system controller, whichis based on the emitted signal detected from the first site; and whereinthe system controller, prior to the array scan, additionally causes theapparatus to execute a pre-scan which includes: scanning theinterrogating light across multiple sites on the array package using alower sensitivity than that used in step (a); and detecting at thedetector, signals from respective scanned sites emitted in response tothe interrogating light; wherein the determination made by the systemcontroller that the emitted signal from the first site during a scan isoutside a predetermined range, is based on the emitted signal obtainedfrom the first site during the pre-scan.
 25. An apparatus forinterrogating an addressable array of multiple features of differentmoieties, carried by an array package, comprising: (a) a light systemwhich provides an interrogating light of variable power in response to acontrol signal characteristic; (b) a scanning system which scans theinterrogating light across multiple sites on the array package, whichscanned sites include multiple features of the array; (c) a signaldetector which detects signals from respective sites emitted in responseto the interrogating light; and (d) a system controller which calibratesinterrogating light power in response to the control signalcharacteristic, and which adjusts the interrogating light power for afirst site on the array package during the array scan using thecalibration, based on location of the first site or on a determinationthat the emitted signal from the first site will be outside apredetermined range; wherein the controller repeats the calibrationprior to each scanning of an array package.
 26. An apparatus forinterrogating an addressable array of multiple features of differentmoieties, carried by an array package, comprising: (a) a light systemwhich provides an interrogating light of variable power in response to acontrol signal characteristic; (b) a scanning system which scans theinterrogating light across multiple sites on the array package, whichscanned sites include multiple features of the array; (c) a signaldetector which detects signals from respective sites emitted in responseto the interrogating light; and (d) a system controller which calibratesinterrogating light power in response to the control signalcharacteristic, and which adjusts the interrogating light power for afirst site on the array package during the array scan using thecalibration, based on location of the first site.
 27. A computer programproduct for use on an apparatus for interrogating an addressable arrayof multiple features of different moieties by means of an interrogatinglight generated from a variable optical attenuator through which lightfrom a light source has passed, and which optical attenuator isresponsive to a control signal to alter the power of the interrogatinglight, the program product comprising: a computer readable storagemedium having a computer program stored thereon which, when loaded intoa computer of the apparatus performs the steps of: (a) causing theinterrogating light to scan across the addressable array; (b) receivingan indication of interrogating light power from the variable opticalattenuator; and (d) adjusting the attenuator control signal to alterinterrogating light power, based on the detected power.
 28. A computerprogram product according to claim 27 wherein the attenuator controlsignal is adjusted to decrease light power when the interrogating lighthas scanned off the array.
 29. A computer program product for use on anapparatus for interrogating an addressable array of multiple features ofdifferent moieties by means of an interrogating light generated from alight system which provides an interrogating light of variable power inresponse to a control signal, the program product comprising: a computerreadable storage medium having a computer program stored thereon which,when loaded into a computer of the apparatus performs the steps of: (a)causing the interrogating light to scan across multiple sites on anarray package including an addressable array of multiple features ofdifferent moieties, which scanned sites include multiple features of thearray; and (b) adjusting the control signal to alter the interrogatinglight power for a first site on the array package during the array scan,based on location of the first site or on a determination that theemitted signal from the first site will be outside a predeterminedrange.
 30. A computer program product according to claim 29 wherein theinterrogating light power is reduced based on a determination that theemitted signal from the first site will exceed a predetermined value.31. A computer program product according to claim 29 wherein theadjustment is based on location of the first site.
 32. A computerprogram product according to claim 29 wherein the determination is basedon an emitted signal detected from the first site.